ABSTRACT The treatment of severe burns relies on autologous skin grafts, which are limited due to donor-site unavailability and substantial comorbidities. Current therapies are unable to eliminate substantial healing defects in severe burns, underscoring the urgent needs of more effective therapies. Our long-term goals are to develop better therapies for severe burns. Mesenchymal stem cells (MSCs) possess regenerative power for burn wounds. Adipose-tissue derived microvascular fragments (MVFs) are native vascularization units and a rich source of the MSCs, endothelial cells, perivascular cells, and adipocytes essential in rebuilding burn- destroyed skin. MVFs provide a better niche for MSCs, maximizing MSC regenerative power. MVFs are also easily isolated from fat tissue collected via minimally invasive liposuction. We have identified regenerative lipid mediators (ReLiMs) that increase MSC or MVF proliferation, survival, and production of growth factors, and promote tissue regeneration. ReLiM1 restored impaired vascularization and healing. However, ReLiMs have short half-lives in wounds, limiting their utility for healing burns and functionalizing MSCs. To address this problem, we developed a hydrogel that can sustain ReLiM levels in burn wounds. ReLiM release was sustained from the hydrogel of biodegradable, non-toxic amino acid-based poly(ester amide) protein-mimic polymers (AA-PEAs). The integration of a sustained-release ReLiM with an AA-PEA-hydrogel (Agel) matrix accelerated healing and promoted MSC/MVF functions. We hypothesize that sustained release ReLiMs combined with Agel scaffolds directly promote healing of severe burns and protect and guide MVF regenerative functions. Our objective is to develop ReLiM-impregnated Agels that deliver ReLiMs and provide scaffolds for healing severe burns and protecting and guiding MVF regenerative functions for better healing as well as to decipher the mechanisms via which this occurs. Aim 1. We will develop the Agel to 1A) sustain ReLiM release and to 1B) provide an optimal ReLiM-releasing biomimetic matrix for skin cell homing, vascularization, and re-epithelization, as well as for the reduction of scarring and increasing wound breaking strength. Aim 2. 2A) We will develop a construct that integrates uncultured autologous MVFs, sustained release ReLiMs, and Agel scaffolds to maximize the healing of deep burns. We predict that the achieved construct protects and guides MVF functions. We will verify results using human MVFs in vitro for better translational value. 2B) We will decipher mechanisms for efficacy of the best MVF-ReLiM-Agel. Impact: This project will provide 1) a ReLiM-Agel dressing that delivers ReLiMs and provides a matrix and covering for efficient healing, 2) a ReLiM- and MVF-carrying Agel matrix that protects and guides uncultured MVFs for more efficient healing, and 3) the underlying mechanistic knowledge. These regenerative lipid mediator functionalized dressings with nonexistent or minimal graft-donor requirements are promising adjuvant therapy to overcome the drawbacks of grafting methods or skin substitutes currently used to treat severe burns.